High-throughput screening system originates from pharmaceutical screening studies, which mainly use 96 or 384 well plate as the reactor array to distribute fluids, and mix sample/reagent with the help of automatic robots. It can realize a throughput of at least 10,000 assays per day through analysis and processing of test results with testing devices and data processing software with high sensitivity and high speed. Due to its powerful screening and analyzing capability, high-throughput screening technique has been further applied to numerous scientific areas, such as biology, medical science and chemistry. However, multiple-well plate based commercial high-throughput fluid processing and screening system is increasingly confronted with an enormous challenge accompanied by rapid increase in new targets and samples. Chemical compounds used for screening are mainly obtained from artificial synthesis or separation and purification of natural products, which may result in high cost. Currently, consumption volume of samples for multiple-well plate based high-throughput screening system is between 1 and 100 microliters. It will be possible to reduce the cost by 1,000-100,000 times if it is able to screen samples through manipulating fluids on the picoliter or nanoliter scale. Therefore, most of the studies in both industrial and academic fields focus on miniaturization of high-throughput screening systems. For instance, the minimum fluid processing volume of OryxNano series fluid processing device as developed by British Douglas company is 100 nanoliters (http://www.douglas.co.uk/oryxnano.htm); whereas that of Mosquito series fluid processing device as developed by British TTPLabTech company is down to 25 nanoliters (www.ttplabtech.com/products/mosquito/). Application of such instruments has significantly reduced the screening and R&D cost. Nevertheless, presently there still a lack of techniques and devices for manipulation and high-throughput screening of fluids at several nanoliters or picoliters scales.
Difficulties in performing high-throughput screening at picoliter scale are mainly reflected on the following aspects: 1) existing instruments are unlikely to realize reliable manipulations of fluids at picoliter scale, such as accurate metering and handling of fluids and mixing of sample/reagent; 2) evaporation effect is to be significantly with the reduction of fluid volume; for instance, aqueous phase droplet of one picoliter is to be thoroughly evaporated within 1 second under the typical laboratory conditions; 3) since the fluid in the micro system has an extremely large specific surface area, molecular self-assembly or nonspecific interactions at water/air interface and water/solid interface may result in inactivation, loss, and cross contamination to bioactive molecules, leading to false positive or negative of screening results.
The droplet based microfluidic technique serves as one of active areas for high-throughput screening miniaturization studies. It aims to realize massive generation of droplet micro-reactor of water-in-oil or oil-in-water types, mixing and reaction of samples as well as analysis and verification through control of multi-phase fluids microchannel at micron scale. The volumes of droplet rectors can be flexibly adjusted at picoliter and nanoliter scale, which makes it possible to realize high-throughput screening with extremely low consumption. Evaporation and dilution of solvents inside the droplet reactor as well as the cross contamination among samples can be effectively minimized due to the protection of the oil phase. Self-assembly effect of biologically compatible surfactant on the droplet-oil surface can provide a mild and uniform microenvironment for biochemical screening and reaction, which is favorable for improving the accuracy of analysis and screening. Meanwhile, limited volume of a droplet reactor can also accelerate the mass transfer, and improve reaction efficiency. Therefore, it is possible that the droplet-based microfluidic technique may become a new generation of high-throughput screening technique due to its excellent properties.
Presently, there are three droplet based microfluidic screening methods, namely droplet cartridge method, slipchip method and droplet assembling method. In droplet cartridge method, first, droplet capillary loads samples to be screened to the capillary to form droplet through sequential aspiration. Second, the capillary is connected to the channel of a microfluidic chip to inject target solution into the droplet for reaction via a T-shape interface on the chip. Finally, the droplet reactor formed is collected into another capillary for incubation reaction and testing (Zheng B., Ismagilov R. F. Angew. Chem., Int. Ed., 2005, 44, 2520). According to the Slipchip method, samples to be screened are loaded into the groove array on the lower chip to form droplet. After that, the upper chip is slid to mix the target reagent solution inside the channel on the upper chip with droplet on the lower chip for triggering reaction and screening (Du W. B., Li L., Nichols K. P., Ismagilov R. F. LabChip, 2009, 9, 2286). However, aforesaid two methods require manual loading of droplets, connection of capillary and channel on the chip and precise chip sliding, which are unlikely to be applied to screening of samples on a large scale. Droplet assembling method is capable of achieving the mixing of samples to be screened and reagent during the formation of droplets through quick automatic switching between the sample and reagent tube. After that, the droplets are to be stored to the capillaries and chips for reaction and test (Du W. B., Sun M., Gu S. Q., Zhu Y., Fang Q. Anal. Chem., 2010, 82, 9941, Fang Qun, Du Wenbin and Sun Meng, Sequential Droplet Technique Based Microfluidic Droplet Generation System and Its
Application Methods, Chinese Invention Patent, Application No.: 201010250945.). Despite of the fact that sequential droplet assembling technique has solved the problem of automatic screening of samples on a large scale, it is difficult to accelerate the screening process due to the sequential assembling method for generation of droplets containing samples and reagent. Furthermore, due to scale effect as brought forth by miniaturization, aforesaid several droplet screening methods are unlikely to realize biological screening and test in picoliter scale.
There are mainly two methods for parallel addition of reagents into the microfluidic droplet system. The first method uses one T-shape branch channel to inject the same reagent into different droplets in the main channel (Zheng B. Ismagilov R. F. Angew. Chem., Int. Ed., 2005, 44, 2520). Normally, such method is used in combination with aforesaid droplet cartridge method for droplet based micro screening. However, the major problem with this method lies in excessive accumulation of residual droplet samples at the intersection of the T-shape channel, which may result in cross contamination to droplet. Another method makes use of droplet mixing technique for parallel injection of reagents. First, mutually paired sample and reagent droplets are produced in the microchannel; Second, hydrodynamic or dielectric approaches are used to make each paired droplets fused into a single micro reactor (the S Y, Lin R. Hung L. H., Lee A. P., Lab Chip, 2008, 8:198). However, such method is complicated in channel structure and difficult in processing, which is unlikely to be used to the screening system containing a large quantity of different samples. Moreover, aforesaid two methods are unlikely to be realized without many manual adjustments such as complicated flow rate regulation, control of droplet frequency and size, as well as feedback recording of droplet compositions. Therefore, it is unavailable for reliable automation, and thus is difficult for instrument industrialization.